Presence and quantity of rare circulating tumor cells (CTCs) in the bloodstream of cancer patients provide a potentially accessible source for evaluating prognosis and treatment monitoring.Separation of CTCs from peripheral blood,as a ‘‘liquid biopsy,is expected to be able to replace conventional tissue biopsies of metastatic tumors for therapy guidance.In this study,we developed a multistage microfluidic device for the continuous label-free separation and enrichment of CTCs from blood cells based on cell size and deformability.A numerical model is developed to evaluate velocity field fluctuations inside the device,which is validated against the experimental phenomena and serves as a theoretical foundation in optimizing the operating conditions.Using a unique combination of inertial forces and steric hindrance in a microfluidic system,we successfully separated cancer cells (MCF-7,K562 and Hela) spiked into whole blood.The processing parameters of inertial focusing region and hydrodynamic microfluidic railing region were optimized to achieve high throughput and high efficiency separation,comparable to existing CTCs isolation technologies.Results from experiments conducted with cancer cells spiked into the 1% hematocrit (Hct) blood condition indicate >90% cell recovery at the throughput of 2.24×107 cells/min,and the impressive enrichment of cancer cells was >206-fold.Therefore,we suggest that our system driven by purely hydrodynamic forces has practical potential to be applied either alone or as a sample preparation platform for use in fundamental studies and clinical diagnosis,thus paving the way for the development of a novel passive microfluidic device for future cancer research.